Atherosclerotic cardiovascular disease remains the leading killer of men and wormen in the United States, especially in persons with diabetes, hypertension, and renal disease. Since treating humans in the late stages of atherosclerosis with vitamins C and E shows little benefit, this project targets the role of these antioxidant vitamins in the early stages of the process in cells and in mice prone to atherosclerosis. The rationale for this approach is the demonstration almost 50 years ago that moderate vitamin C deficiency in guinea pigs causes lesions indistinguishable from those of early human atherosclerosis. The first aim will evaluate vitamin C function in cell culture models of the three main cells involved in the atherosclerotic process: endothelial cells, vascular smooth muscle cells, and macrophages. Although antioxidant functions of vitamin C and interactions with vitamin E will be addressed, emphasis will be placed on how stimulation of collagen synthesis by vitamin C affects endothelial and smooth muscle cell proliferation and differentiation, and on how the vitamin can modify key macrophage functions as these relate to atherosclerosis. The second aim will utilize the ApoE-deficient mouse model of atherosclerosisto assess whether varying amounts intracellular vitamin C affect the progression, severity, and nature of the atherosclerotic lesions. Key to this aim is the use of mice that lack the ability to synthesize vitamin C in concert with mice that lack cellular transport of the vitamin. For example, using fetal liver cell transplants in lethally irradiated mice, it will be possible to test whether selective deficiency of vitamin C in macrophages lacking vitamin C transport worsens the atherosclerotic process. In the third aim, macrophages prepared from these animals will be cultured and used to define the role of differing intracellular vitamin C concentrations on macrophage function, and on antioxidant interactions with vitamin E. By focusing on mechanisms in cell models and on early disease in animal models, this project will show whether and how vitamin C prevents atherosclerosis.